Diabetes is a disease arising from shortage of blood insulin and impaired insulin function, resulting in increased blood glucose concentrations, accompanied by complications such as neuropathy, visual disturbance, renal damage, etc. About 7 million people are afflicted with diabetes in Japan alone. There are two major types of diabetes: Type 1 diabetes is caused by an autoimmune destruction of insulin-producing pancreatic β cells, resulting in an absolute lack of insulin. On the other hand, type 2 diabetes is caused by the expression of insulin resistance in target tissues, such as muscle, fat, and liver, or by a decrease in blood insulin levels due to a decline in pancreatic β cell function. Thus, it can be said that both type 1 and type 2 result from impaired pancreatic β-cell function.
For treatment of type 1 diabetes, insulin injections are conventionally used to lower blood glucose levels in most cases. It is needless to say that, in such cases, four injections a day are laborious to patients. For treatment of type 2, the PPARγ inhibitor, which reduces insulin resistance, is used in some cases. However, the inhibitor is not very effective and causes obesity as a side effect, as has been pointed out. In other cases, agents such as sulfonylurea, which promotes insulin release from β cells, are used, but they are also not very effective.
Apart from drug dependent treatment, a new treatment by transplantation of cells or tissue has been considered promising as regenerative therapy. If large amounts of cells with insulin-releasing ability can be transplanted into type I diabetes patients, four insulin injections per day can be avoided for a long term. Meanwhile, transplantation of insulin-releasing cells is also effective for type 2 diabetes because, irrespective of insulin resistance in target tissues, normal insulin production suppresses the increase of blood glucose level. It is expected that the efficacy of transplantation is far superior to that of treatment with agents currently used, e.g., sulfonylurea, that induce insulin release from β cells.
Porcine pancreatic cells are expected to be utilized in regenerative therapy for diabetes because of their ease of availability, immunological properties, etc. The technique for collecting large amounts of cells and inducing them to differentiate into insulin producing and releasing cells to a sufficient degree has never been known in any cell system.
Most of pancreatic β cells are generated in the fetal period and then proliferate and differentiate very slowly (Herrera, P. L. et al., Development 127: 2317-2322 (2000)). However, experiments have shown that when the pancreas is damaged β cells actively differentiate and proliferate. Differentiation and proliferation of β cells, together with growth of remnant β cells, occur from pancreatic ductal cells both in adult mice subjected to a 90% partial pancreatectomy and in mice that have developed impaired glucose tolerance due to β cell destruction induced by alloxan. These findings have revealed that stem cells are also present in the adult pancreas and have regenerative capacity (Bonner-Wier S. et al., Diabetes 42: 1715-1720 (2000)). Thus, experiments were performed in which stem cells were isolated from the pancreatic ducts and induced to differentiate into insulin-producing cells (Ramiya, V. K. et al., Nature Med. 6: 278-282 (2000) and Bonner-Weir, S. et al., Proc. Nat. Acad. Sci. USA 97: 7999-800 (2000)). Still, a marker of pancreatic stem cells was not known. It was reported last year that nestin, a marker expressed in neural precursor cells, is a marker of pancreatic stem cells, and nestin was confirmed to be expressed in adult pancreatic duct. (Hunziker, E. et al., Biochem. Biophys. Res. Commun. 271: 116-119 (2000)). Furthermore, in vitro culture and differentiation into cells with phenotype including that of insulin-producing cells was succeeded (Zulewski, H. et al., Diabetes 50: 521-533 (2001)). In addition, differentiation of ES cells into pancreatic inlet-like tissues was also succeeded (Lumelysky, N. et al., Science 292: 1389-1394 (2001)). These techniques are expected to be clinically applied in regenerative medicine of the pancreas.
While studies are under way on cells that can potentially serve as materials for pancreatic β cells, physiologically active substances that induce differentiation of pancreatic β cells are considered promising for clinical application in the field of regenerative medicine. Examples of substances that have thus far been known as inducers of differentiation into β cells include activin A, which belongs to the TGF-β superfamily (Demeterco, C. J. et al., Clin. Endo. 85: 3892-3897 (2000)); betacellulin (BTC), which belongs to the EGF family (Ishiyama, N. et al., Diabetologia 41: 623-628 (1998) and Yamamoto, K. et al., Diabetes 49: 2021-2027 (2000); hepatocyte growth factor (HGF) (Ocana, A. G. et al., J. Bio. Chem. 275: 1226-1232 (2000)); basic fibroblast growth factor (bFGF) (Assady, S. et al., Diabetes 50: 1691-1697 (2001)); etc. These substances are proteins, which are not suitable for oral administration. Further, it is difficult to introduce such substances by means of injection because of their immunological problem and instability.
Meanwhile, it has been reported that among low-molecular-weight compounds, nicotinamide acts as a poly (ADP-ribose) synthetase inhibitor, promoting regeneration of pancreatic β cells (Watanabe, T. et al., Proc. Natl. Acad. Sci. USA 91: 3589-3592 (1994) and Sjoholm, A. et al., Endocrinology 135: 1559-1565 (1994)). In addition, nicotinamide has also been reported to promote the expression of Reg protein (Watanabe, T. et al., Proc. Natl. Acad. Sci. USA 91: 3589-3592 (1994)), which promotes the proliferation and differentiation of pancreatic β cells (Akiyama, T. et al., Proc. Natl. Acad. Sci. USA 98: 48-53 (2001)). In another report, fetal porcine pancreatic islet-like cell clusters (ICCs) were induced to differentiate into insulin-producing cells using sodium butyrate and dexamethasone (Korsgren, O. et al., Ups J. Med. Sci. 98: 39-52 (1993)), but the technology is less likely to be put into practical use because of the low specificity.
Meanwhile, the structures of conophylline (Umezawa, K. et al., Anticancer Res. 14: 2413-2418 (1994)) and conophyllidine (Kam, T. S. et al., J. Nat. Prod. 56: 1865-1871 (1993)), both of which are alkaloids isolated from leaves of an Apocynaceae family plant grown in Malaysia and Thailand are known, as shown in FIG. 1. Conophylline is known to exhibit anti-tumor activity in animals (Umezawa, K. et al., Drugs Exptl. Clin. Res. 22: 35-40 (1996)).
It is also known that conophylline induces insulin production of pancreatic acinar carcinoma AR42 J-B13 cells. (The Book of Abstracts (01-21) of the 45th Annual Meeting of the Japan Diabetes Society held in Tokyo, May 18, 2002). However, these cancer cells did not release insulin into culture medium.
The object of the present invention is to provide agents capable of inducing insulin production and/or secretion of non-neoplastic cells derived from the pancreas.